CN101012453A - CHO cell strain for highly effective expressing rhBMP2 and establishing method thereof - Google Patents

Abstract

The invention relates to constructing a CHO cell strain capable of highly expressing exogenous rhBMP2. Mainly use dihydrofolate reductase-deficient Chinese hamster ovary cells (CHO- ^dhfr- ) as host cells, stably transfect exogenous hBMP2 plasmids, and obtain stable and high-efficiency expression of rhBMP2 after drug resistance screening and gene amplification cell line. The secreted expression product of the cell line can efficiently induce the expression of alkaline phosphatase in the myoblast cell line C2C12 to show its activity of stimulating osteogenesis. The CHO (rhBMP-2) monoclonal cell line was obtained, and the expression level of BMP2 was up to 7.83ug/24h·10 ⁶ cells. The rhBMP2 expressed by the recombinant CHO cell strain established by the method has similar physical and chemical properties and biological activities to natural human BMP2, and can be used for treating clinical diseases such as bone defect, nonunion and delayed fracture healing caused by different reasons.

Description

CHO cell line highly expressing rhBMP2 and its establishment method

technical field

The invention belongs to the technical field of genetic engineering pharmacy, and relates to a recombinant CHO cell CHO (rhBMP-2) highly expressing human bone morphogenetic protein 2 and its establishment method. Specifically, an immortalized mammalian cell line that is stably transfected with the coding sequence of the human bone morphogenetic protein 2 (hBMP2) gene is constructed, and a cloning cell line that stably and efficiently expresses rhBMP2 is obtained after pressure selection and gene amplification.

Background technique

Due to trauma, infection, tumor and dysplasia, the bone loses some bone quality, and the symptoms such as refractory bone defect, nonunion and delayed fracture union are one of the most difficult problems in clinical practice. The best way to treat bone defects is autologous bone grafting, but in addition to the limited source and the possibility of complications in the bone harvesting area, the repair speed of autologous bone grafting is relatively slow, which greatly limits its widespread use. Allograft bone transplantation is naturally not a problem. It has been the goal of many laboratories at home and abroad to find the factors that promote bone growth and mix them with suitable bone graft substitute materials to treat clinical bone defects, nonunions and delayed union of fractures caused by different reasons. Obtaining a large amount of stable and biologically active human bone morphogenetic protein 2 in vitro has been a very active research field at home and abroad.

Currently, most of the bone graft substitute materials commonly used in clinical practice have no osteoinductive activity, and are only artificial or natural materials with osteoconduction and support functions. If these materials are combined with bone morphogenetic protein (BMP), an osteogenic factor with high osteoinductive activity, according to the principle of tissue engineering, it will not only have osteoinductive activity, but also have osteoconductive and supporting functions, which can greatly improve bone health. The clinical practical value of graft repair materials.

In 1889, Senn found that decalcified bone matrix can promote the repair of bone defects. In 1930, Levander injected the ethanol crude extract of bone into mouse tissue and found that it could induce new bone formation. In 1961, Sharrard and Collins used ethylenediaminetetraacetic acid decalcified autogenous bone for the treatment of spinal fusion in children. In 1965, Urist found an active substance that could induce bone formation in the bone matrix, and named this active substance "bone morphogenetic protein". In 1983, Reddi and Sampath confirmed that it was the protein components in the bone matrix rather than the bone matrix itself that had the activity of inducing bone formation. In 1988, Johnson and his colleagues conducted a clinical trial using purified human BMP to treat bone defects.

At present, more than ten bone morphogenetic proteins have been found, among which bone morphogenetic proteins 2, 6, and 7 have strong osteoinductive effects. In particular, BMP-2 has strong osteogenic activity and can induce bone formation in vivo alone, so it has been used in many clinical applications such as fracture healing, spinal fusion, plastic surgery, and dental tissue engineering.

The homology between different bone morphogenetic protein molecules is very high, and they are all synthesized in the form of a nitrogen-terminal signal peptide, propeptide and carbon-terminal mature peptide precursor. After processing and modification, the signal peptide and propeptide are cut off, and mature The peptide moieties form glycosylated homo- or heterodimers, which induce bone formation. However, natural BMP has low tissue content, limited sources, cumbersome separation and purification steps, high cost, and adverse reactions such as infection and allergy, so it is difficult to meet the needs of clinical practice.

The establishment of gene recombination technology has opened up a new way for the production of therapeutic proteins. Now, human BMP can be prepared in large quantities through gene recombination technology, and it can be used for the treatment of related diseases such as clinical bone defects. Due to the limitations of prokaryotic exogenous gene expression systems, eukaryotic exogenous gene expression systems are widely used to express biologically active proteins that require post-translational modification, especially mammalian expression systems have accurate post-transcriptional modification functions, Compared with prokaryotic cell expression system and yeast cell expression system, the expressed glycosylated protein is closest to the natural protein molecule in terms of molecular structure, physical and chemical properties and biological function, and is currently the preferred system for recombinant glycoprotein production.

Among the different mammalian cell expression systems, the Chinese hamster ovary cell (CHO) exogenous protein expression system has become the most successful mammalian cell line for exogenous protein expression after years of practice. Its advantage is that the exogenous gene can be stably It is integrated in the chromosome of the cell and is easy to be cultured on a large scale. These two characteristics create conditions for the large-scale preparation of exogenous proteins. Among them, dihydrofolate reductase (DHFR)-deficient CHO cells lack endogenous dihydrofolate reductase, and when the expression vector containing dihydrofolate reductase gene and target gene is co-transfected, the The gene and the target gene are usually integrated at the same site on the host chromosome. In the presence of the competitive inhibitor of DHFR: methylpterin (MTX), the increase in the copy number of the DHFR gene drives the increase in the expression of the target gene. .

At present, the search results of relevant Chinese patent data show that by co-transfecting human BMP2 and DHFR genes into DHFR-deficient CHO cells, after screening and amplification, there have been no reports of recombinant CHO cell lines that efficiently and stably express human BMP2.

Contents of the invention

The purpose of the present invention is mainly to provide a recombinant CHO cell line CHO (rhBMP-2) capable of highly expressing human bone morphogenetic protein 2.

Another object of the present invention is to establish a technical platform that may be applied to express other biologically active proteins by establishing a recombinant CHO (rhBMP-2) cell line.

Another object of the present invention is to disclose the application of the recombinant CHO cell line CHO (rhBMP-2) in the preparation and treatment of inducing bone formation activity and promoting bone defect repair. In particular, the expressed recombinant CHO cell line CHO (rhBMP-2) has been proved to have obvious therapeutic effects in inducing bone formation in vitro, inducing ectopic bone formation in vivo and promoting bone defect repair.

The technical solution adopted by the present invention to solve its technical problems is:

A recombinant CHO cell strain highly expressing recombinant human bone morphogenetic protein 2, named CHO (rhBMP-2), this cell strain has been preserved by the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microorganisms, and the preservation time was September 2006 December 12. Classified and named Chinese hamster ovary cell line, the preservation number is CGMCC NO.1813

The recombinant CHO cell strain highly expressing recombinant human bone morphogenetic protein 2 according to the present invention is characterized in that it mainly uses dihydrofolate reductase-deficient Chinese hamster ovary cells (CHO-dhfr ^- ) as host cells, and after screening, After amplification, stable and high-efficiency expression cell lines were obtained. The secreted expression product of the cell line can efficiently induce the expression of alkaline phosphatase in the myoblast cell line C2C12, and obtain the CHO (rhBMP-2) monoclonal cell line, and its expression level reaches 7.8 3ug/24h·10 ⁶ cells.

The expression vector of the present invention is not limited to a specific expression vector, as long as it can recombine with the DNA fragment to form a suitable expression plasmid. Such as prokaryotic exogenous gene expression system; eukaryotic exogenous gene expression system; yeast cell expression system, etc., the preferred expression vector is eukaryotic exogenous gene expression system; it can be recombined with the DNA fragment and is easy to synthesize , can be effectively secreted into the culture medium. And it can form expression plasmid suitable for expressing active protein.

The construction method of the recombinant CHO cell line CHO (rhBMP-2) of recombinant human bone morphogenetic protein 2 of the present invention comprises the following steps:

(1) Artificial oligonucleotide synthesis and polymerase chain amplification technology to clone the cDNA of the full-length sequence of human BMP2.

(2) The cDNA of the full-length sequence of human BMP2 was cloned into the eukaryotic expression vector pCDNA3.1(+), and the mammalian cell expression vector pCDNA3.1(+)-BMP2 was constructed.

(3) pCDNA3.1(+)-BMP2 and pSV2-dhfr were co-transfected into CHO-dhfr ^- with lipofectine2000.

(4) The stable transfected cell lines integrating BMP2 and dhfr genes were screened with G418 and the selection medium of hypoxanthine, thymine and glycine.

(5) The method of gradually increasing the concentration of methotrexate to amplify the copy number of the target gene.

(6) Western Blot was used to detect rhBMP2 in the culture medium of recombinant CHO cells.

(7) Single clones were isolated from mixed clones by limiting dilution method, and the expression level of rhBMP2 in the culture medium of the single clones was detected by ELISA method, and the engineered cell lines that highly express rhBMP2 were screened.

(8) The engineered cell line secretes and expresses rhBMP2 in the medium during the culture process.

Concrete preparation method of the present invention is described as follows:

1. Test materials and methods

1.1. Cell culture

Dihydrofolate reductase-deficient Chinese hamster ovary cells CHO- ^dhfr- (CRL-9096; ATCC) were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, cultured in IMDM medium (GIBCO), and added 10 ^-4 mol/L hypoxanthine and 1.6×10 ^-5 mol/L thymidine (both purchased from Sigma Company) and 10% dialyzed fetal bovine serum (purchased from Tianjin Haoyang Biological Products Co., Ltd.) . The cells involved in this experiment were all cultured in a cell incubator containing 5% CO ₂ at 37°C.

1.2 Construction of eukaryotic expression plasmids:

Using the previously constructed plasmid pCDNA6A-hBMP-2 containing the full-length sequence of hBMP-2 cDNA (GenBank accession No. NM001200) in our laboratory as a template, the cDNA of the full-length sequence hBMP-2 was amplified by PCR reaction, and the It is connected to the expression vector plasmid pCDNA3.1(+), and the eukaryotic expression plasmid pCDNA3.1(+)-hBMP-2 is constructed. To facilitate cloning, BamHI and XhoI restriction sites were introduced into the upstream and downstream primers respectively.

Upstream primer: 5'-GCTGGATCCACCATGGTGGCCGGGACCCGCTGTC-3'

Downstream primer: 5'-GCGTCTCGAGCTAGCGACACCCACAACCCTCCAC-3'

The product of the ligation reaction was transformed into competent cells E.coli DH5α, and the positive colonies were screened by colony PCR, and the plasmid DNA was further extracted for enzyme digestion identification. The identification results were in line with expectations, and the sequencing results showed that the reading frame was completely correct.

1.3. Transfection and screening of positive clones

The constructed plasmids pCDNA3.1(+)-hBMP-2 and pSV2-dhfr were mixed according to a molar ratio of 5:1, and Lipofectamine2000 (purchased from Invitrogen) was used to co-transfect CHO-dhfr- cells, while empty plasmid pCDNA3. 1(+) and pSV2-dhfr were co-transfected into CHO-dhfr- cells as a control. After 24 hours of cell transfection, the fresh IMDM selection medium (containing 10% FBS, 700 μg/ml G418) was replaced for culture, and the medium was changed every three days until the formation of cell clones.

1.4. Pressurized culture of methotrexate:

All the clones formed in step 1.3 were digested with the cell digestion solution Trypsin-EDTA (purchased from GIBCO), and inoculated into cell culture dishes for mixed culture. After the cells grew to a density of 90%, they were inoculated into new cells at a ratio of 1:6. In the cell culture dish, methotrexate (MTX) (purchased from Sigma Company) was added to the selection medium to make the final concentration reach 0.1 μmol/L, and resistant clones could be seen after about 2 weeks, and all clones were After digestion, mixed culture, after the cells grow normally, the transformant is amplified with 1 μmol/L MTX in the same procedure, and finally a mixed clone of recombinant CHO cells that can grow normally in 1 μmol/L MTX is obtained, named is CHO(rhBMP-2).

1.5. Western blot identification of rhBMP-2

CHO (rhBMP-2) was inoculated into a cell culture dish, and after the cells reached a density of 90%, they were cultured in a serum-free medium (without MTX), and the cell culture medium was collected 2 days later. Proteins in cell culture medium were extracted by trichloroacetic acid precipitation. The extracted protein was analyzed and identified by Tris-Tricine polyacrylamide gel electrophoresis and Western blot. The samples were mixed with DTT-containing and DTT-free loading buffers, electrophoresed on 8% polyacrylamide gels with Rainbow Marker, and then transferred to nitrocellulose membranes with 5% skimmed milk TBST buffer to block overnight. Incubate with primary anti-MP-2 (purchased from R&D Company) monoclonal antibody and secondary antibody anti-mouse IgG (purchased from Promega Company) with horseradish peroxidase for 2 h, then react with ECL reagent and expose .

1.6. Expression level detection of rhBMP-2

According to the method described in "Tissue Culture and Molecular Cytological Techniques", the mixed clones of CHO (rhBMP-2) cells were isolated and cultured, and 14 monoclonal cell lines were obtained. The 14 monoclonal cell lines were inoculated into 96-well plates, and the fresh medium was replaced after the cell growth reached 90% density. After 24 hours, the culture supernatant was collected, and the hBMP-2ELISA Kit (purchased from Wuhan Boster Company) was used to detect the culture medium. The concentration of rhBMP-2 in the supernatant of the liquid was measured, and the number of cells in each well was measured with a CCK-8 kit (purchased from Japan Tongjin Chemical Research Institute), and the expression level of rhBMP-2 of each monoclonal cell line was calculated according to the results.

1.7. Biological activity detection of rhBMP-2

It has been reported in the literature that the mouse myoblast cell line C2C12 differentiates from myoblasts into osteoblasts under the stimulation of BMP-2, and alkaline phosphatase (ALP) is a marker enzyme of osteoblasts. We stimulated and cultured C2C12 with different concentrations of rhBMP-2 conditioned medium, and analyzed the biological activity of rhBMP-2 by measuring the activity of ALP.

1.7.1 Preparation of conditioned medium

CHO (rhBMP-2) was inoculated in IMDM medium containing 10% FBS and 1 μmol/L MTX, and after the cell growth reached 90% density, it was cultured in serum-free DMEM (without MTX), and collected after 24 hours Cell culture medium, 4000g, centrifuge at 4°C for 5min, take the supernatant and put it in a 1.5ml centrifuge tube, store at -70°C, and mix it with normal medium in proportion to use.

1.7.2 Determination of alkaline phosphatase activity:

Add the collected CHO (rhBMP-2) culture supernatant to DMEM at a ratio of 5%, 10% and 20%, respectively, and add FBS at the same time, so that the final concentration of FBS reaches 5%, and culture in this mixed medium C2C12 cells, and the C2C12 cells were cultured in DMEM containing only 5% FBS as a control, and the medium was changed every two days. After stimulating culture for 5 days, first detect the O.D value of each well at 450nm with CCK-8 reagent, then wash 3 times with PBS buffer, add 50μl alkaline phosphatase reaction substrate solution (4 pieces of Sigma#104phosphatasesubstrate dissolved in 15mL 50mmol/L glycine, 1mmol/L MgCl2, pH10.5), incubate at room temperature for 20min. The O.D value was measured at a wavelength of 405nm with a Microplate Reader, and the result was divided by the O.D value measured using the CCK-8 reagent to normalize the ALP result. The average value of 6 replicate wells for each group of samples was plotted.

2. Results

2.1 Construction and identification of rhBMP-2 eukaryotic expression vector:

Using plasmid pCDNA6A-hBMP-2 as a template, carry out PCR reaction to amplify hBMP-2 cDNA, and then insert the PCR product into plasmid pCDNA3.1(+) to obtain hBMP-2 eukaryotic expression vector pCDNA3.1(+) - hBMP-2 (Figure 1, Fig 1). The extracted plasmid was digested with restriction endonuclease and electrophoresed to obtain a fragment of about 1.2kb/5.4kb in size, which was in line with the expected result (Figure 2, Fig2). The sequencing results confirmed the base sequence and reading code of the inserted fragment The box is exactly right.

2.2 Western blot identification of rhBMP-2 expression in CHO cells:

CHO (rhBMP-2) cells were inoculated in a cell culture dish, and after the cells reached 90% density, they were replaced with serum-free DMEM to continue culturing. After 48 hours, the culture medium was collected, and the protein in the culture medium was extracted by trichloroacetic acid precipitation. The extracted protein samples were mixed with loading buffer containing DTT and DTT-free, and identified by Tris-Tricine electrophoresis and Western blot (Fig. 3, Fig3). The results showed that two specific protein bands with a size of about 18 kDa and a size of about 30 kDa appeared in the reduced and non-reduced sample lanes respectively, indicating that CHO (rhBMP-2) secreted and expressed rhBMP-2 protein. According to the calculation of the amino acid composition of hBMP-2, the size of the rhBMP-2 protein without post-translational modification is about 13kDa, so it is speculated that the expressed rhBMP-2 is post-translational modified by glycosylation and forms a homodimer secreted into the culture medium.

2.3 Detection of rhBMP-2 expression

14 monoclonal cell lines of rCHO (hBMP-2) were obtained by the method of single cell isolation and culture, and the expression level was detected by ELISA method. The results showed that the expression level could reach 3.6-7.83μg/24h/106cells.

2.4 Detection of biological activity of rhBMP-2

C2C12 cells were stimulated with conditioned media containing 5%, 10% and 20% CHO (rhBMP-2) cell culture medium for 24 hours, and C2C12 cells were cultured in DMEM containing only 5% FBS as a control, and alkaline phosphate was detected after 5 days Enzyme activity (Figure 4, Fig4). The results showed that the alkaline phosphatase activity in the C2C12 cells cultured in the conditioned medium made of CHO (rhBMP-2) cell culture medium was significantly higher than that of the control group, and showed an obvious dose-dependent manner, which indicated that the expression rhBMP-2 has strong biological activity. The beneficial effects of the present invention are:

On the basis of comprehensive analysis of previous studies on bone morphogenetic protein 2, using dihydrofolate reductase-deficient Chinese hamster ovary cells as an expression system, an engineering cell line that stably and efficiently expresses recombinant human BMP2 was established through gene recombination technology . And the activity test proves that the recombinant human BMP2 secreted and expressed by the recombinant CHO cell line has significant osteogenic activity, the recombinant human BMP2. The rhBMP2 expressed by the recombinant CHO cell strain established by the method has physical and chemical properties and biological activities more similar to natural human BMP2, and can be used to treat clinical diseases such as bone defect, nonunion and delayed fracture union caused by different reasons.

Description of drawings

Figure 1 Flow chart of the construction of hBMP-2 expression vector pcDNA3.1(+)-hBMP-2.

Figure 2 Construction verification of pCDNA3.1(+)-BMP2. Among them A: hBMP2 D: pCDNA3.1(+)-BMP2 digestion identification.

Figure 3 Western Blot identification of recombinant CHO cells expressing rhBMP2. Where M is the molecular weight; 1 is the control; 2 is the non-reducing condition; 3 is the reducing condition.

Fig. 4 Biological activity analysis of recombinant human BMP2. Wherein ALP/CCK-8: relative activity of alkaline phosphatase rhBMP2 CM: CHO (BMP2) conditioned medium.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings of the description and the following specific examples. The examples are for illustration only, and are by no means intended to limit the scope of the present invention in any way.

Embodiment 1: Construction of pCDNA3.1(+)-BMP2

According to the gene sequence of human bone morphogenetic protein 2 (BMP2, SEQ ID NO.1), two pairs of specific primers (B2F, B2R) were designed and synthesized. Using B2F and B2R as primers, a fragment with a size of about 1200 bp was amplified by polymerase chain reaction ( FIG. 2A ). This fragment was cloned into a eukaryotic expression vector to obtain pCDNA3.1(+)-BMP2, which was confirmed to be consistent with the expected target sequence by restriction enzyme digestion (Fig. 2B) and sequencing.

B2F: 5'-gctggatccaccatggtggccgggacccgctgtc-3'

B2R: 5'-gcgtctcgagctagcgacacccacaaccctccac-3'

Example 2: Establishment of engineering cell strains stably and efficiently expressing recombinant human BMP2

Dihydrofolate reductase-deficient Chinese hamster ovary cells (CHO-dhfr-) use complete medium supplemented with glycine, hypoxanthine, thymine 10% FBS IMEDM (Hyclone), at 37°C, 5% CO2, 10cm culture plate Cells were cultured to about 80% confluency. Use cationic liposome Lipofectine 2000 (Invitrogen) to pCDNA3.1(+)-BMP2 and pSV2-DHFR at a ratio of 10:1, transfect according to the method in the operation manual, and then use the selection method containing 700ug/ml G418 and 10% FCS IMDM Media screened neo and dhfr positive clones. The obtained positive clones were inoculated on a 10cm cell culture dish with 1×10 ⁶ /10ml cells, and the cells were gradually cultured with IMDM medium containing 100nM and 1uM methotrexate (MTX) and 10% FBS to amplify the target gene, and finally The recombinant cells that can grow normally in the medium containing 1uM MTX were obtained. The protein in the cell culture fluid of the recombinant cells was extracted and subjected to Western Blot to detect the expression of rhBMP2 protein in the recombinant cells ( FIG. 3 ). The mixed clones amplified by 1uM MTX were seeded in a 96-well cell culture plate at 0.3-0.5 cells/well, and cultured with 10% FBS DMEM. When the cells were close to confluence, the culture medium was collected, and the engineered cell line rCHO (BMP2) stably and highly expressing rhBMP2 was detected and screened by ELISA.

Example 3: Analysis of bone formation activity induced by recombinant human BMP2 in vitro

When the myoblast cell line C2C12 was cultured with DMEM containing 10% FBS to 70-80% confluence, washed with PBS, digested with trypsin, counted, seeded in 48-well cell culture plate at 2× ¹⁰⁴ cells/well, placed in Incubate in a 37°C, 5% CO2 incubator. After 24 hours, the culture medium was aspirated, washed twice with PBS, and the conditioned medium containing 5%, 10%, and 20% rCHO (BMP2) 24h cell culture solution was added to stimulate the culture of C2C12. After 5 days, count with the CCK-8 kit The number of cells per well, and then the alkaline phosphatase activity was measured by colorimetry (Figure 4), and the expression level can reach up to 7.83 μg/24h/10 ⁶ cells.

Sequence listing:

<110> Nankai University

<120> CHO cell line highly expressing rhBMP2 and its establishment method

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ctgcggtctc ctaaaggtcg accatggtgg ccgggacccg ctgtcttcta gcgttgctgc 360

ttccccaggt cctcctgggc ggcgcggctg gcctcgttcc ggagctgggc cgcaggaagt 420

tcgcggcggc gtcgtcgggc cgcccctcat cccagccctc tgacgaggtc ctgagcgagt 480

tcgagttgcg gctgctcagc atgttcggcc tgaaacagag accaccccc agcagggacg 540

ccgtggtgcc cccctacatg ctagacctgt atcgcaggca ctcaggtcag ccgggctcac 600

ccgccccaga ccaccggttg gagagggcag ccagccgagc caacactgtg cgcagcttcc 660

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tctttaattt aagttctatc cccacggagg agtttatcac ctcagcagag cttcaggttt 780

tccgagaaca gatgcaagat gctttaggaa acaatagcag tttccatcac cgaattaata 840

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gtgggtgtcg ctagtacagc aaaattaaat acataaatat atatata 1547

Claims (5)

1, its preserving number of recombinaant CHO cell strain CHO (rhBMP-2) of efficiently expressing recombinant human Delicious peptide 2 is CGMCC NO.1813.

2, the recombinaant CHO cell strain of efficiently expressing recombinant human Delicious peptide 2 is characterized in that, utilizes Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell (CHO-dhfr ^-) as host cell, the cell strain of after screening, amplification, being stablized, efficiently expressing; It is that C2C12 expresses alkaline phosphatase that the secreting, expressing product of this cell strain can efficiently be induced sarcoplast, obtains the strain of CHO (rhBMP-2) monoclonal cell, and its expression amount reaches 7.83ug/24h10 ⁶Cell.

3, the described recombinaant CHO cell strain of claim 1～2 CHO (rhBMP-2) has the application of inducing aspect the active and promotion bone defect repair of bone forming in preparation treatment.

4, the establishment method of claim 1 or 2 described CHO (rhBMP-2) cell strains may further comprise the steps:

(1) the synthetic and polymerase chain amplification technique of artificial oligonucleotide, the cDNA of human cloning BMP2 full length sequence;

(2) cDNA of people BMP2 full length sequence is cloned among the carrier for expression of eukaryon pCDNA3.1 (+), makes up mammalian cell expression vector pCDNA3.1 (+)-BMP2;

(3) use positively charged ion plasmalogen lipofectine2000 with pCDNA3.1 (+)-BMP2 and pSV2-dhfr cotransfection CHO-dhfr ^-

(4) with the selection substratum screening integration BMP2 of G418 and xanthoglobulin, thymus pyrimidine and glycine and the stable transfected cells strain of dhfr gene;

(5) progressively increase progressively the mode of methotrexate concentration, the copy number of amplifying target genes;

(6) detect rhBMP2 in the recombinaant CHO cell substratum with Westen Blot;

(7) from mix the clone, be separated to mono-clonal by limiting dilution assay, utilize the expression amount of rhBMP2 in the ELISA method detection mono-clonal substratum, screen the engineering cell strain of highly effective expressing rhBMP 2;

(8) in the engineering cell strain culturing process secreting, expressing rhBMP2 in substratum.

5, the recombinaant CHO cell strain CHO (rhBMP2) that expresses of the described methods of claims 4 induces the dystopy bone forming and promotes application aspect the bone defect repair in the external evoked bone forming of preparation treatment, body.

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